pZac2.1 gfaABC1D-BioID2-BioID2-HA

LOCUS       pZac2.1 gfaABC1D-BioID2-BioID2-HA                6630 bp ds-DNA     circular SYN 24-NOV-2021
DEFINITION  Expresses the promiscuous small biotin ligase, BioID2, in 
            astrocytes.
ACCESSION   .
VERSION     .
KEYWORDS    pZac2.1 gfaABC1D-BioID2-BioID2-HA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6630)
  TITLE     Bacterial expression plasmids for production of protein-fused BioID2
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6630)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 24, 2021 from SnapGene Server 1.1.58
            http://www.biofeng.com
FEATURES             Location/Qualifiers
     source          1..6630
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        143..173
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   259..388
                     /label=AAV2 ITR
     intron          1239..1371
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1354..1373
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1416..1434
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1461..2159
                     /codon_start=1
                     /product="promiscuous R40G mutant of a biotin protein 
                     ligase from Aquifex aeolicus (Kim et al., 2016)"
                     /label=BioID2
                     /translation="MFKNLIWLKEVDSTQERLKEWNVSYGTALVADRQTKGRGGLGRKW
                     LSQEGGLYFSFLLNPKEFENLLQLPLVLGLSVSEALEEITEIPFSLKWPNDVYFQEKKV
                     SGVLCELSKDKLIVGIGINVNQREIPEEIKDRATTLYEITGKDWDRKEVLLKVLKRISE
                     NLKKFKEKSFKEFKGKIESKMLYLGEEVKLLGEGKITGKLVGLSEKGGALILTEEGIKE
                     ILSGEFSLRRS"
     CDS             2244..2942
                     /codon_start=1
                     /product="promiscuous R40G mutant of a biotin protein 
                     ligase from Aquifex aeolicus (Kim et al., 2016)"
                     /label=BioID2
                     /translation="MFKNLIWLKEVDSTQERLKEWNVSYGTALVADRQTKGRGGLGRKW
                     LSQEGGLYFSFLLNPKEFENLLQLPLVLGLSVSEALEEITEIPFSLKWPNDVYFQEKKV
                     SGVLCELSKDKLIVGIGINVNQREIPEEIKDRATTLYEITGKDWDRKEVLLKVLKRISE
                     NLKKFKEKSFKEFKGKIESKMLYLGEEVKLLGEGKITGKLVGLSEKGGALILTEEGIKE
                     ILSGEFSLRRS"
     CDS             2952..2978
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    3003..3591
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3056..3076)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3474..3485)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3655..3776
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3666..3685)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3720..3739
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3938..4067
                     /label=AAV2 ITR
     primer_bind     complement(4086..4103)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4086..4102)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4095..4117)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4244..4699
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4331..4350)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4541..4562
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4725..4829
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4830..5690
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5048..5067)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5861..6449
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6350..6369
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6603..6620
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccagattta
      241 attaaggcct taattaggct gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc
      301 gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga
      361 gtggccaact ccatcactag gggttccttg tagttaatga ttaacccgcc atgctactta
      421 tctacgtagc catgctctag gaagatctaa catatcctgg tgtggagtag gggacgctgc
      481 tctgacagag gctcgggggc ctgagctggc tctgtgagct ggggaggagg cagacagcca
      541 ggccttgtct gcaagcagac ctggcagcat tgggctggcc gccccccagg gcctcctctt
      601 catgcccagt gaatgactca ccttggcaca gacacaatgt tcggggtggg cacagtgcct
      661 gcttcccgcc gcaccccagc ccccctcaaa tgccttccga gaagcccatt gagcaggggg
      721 cttgcattgc accccagcct gacagcctgg catcttggga taaaagcagc acagccccct
      781 aggggctgcc cttgctgtgt ggcgccaccg gcggtggaga acaaggctct attcagcctg
      841 tgcccaggaa aggggatcag gggatgccca ggcatggaca gtgggtggca gggggggaga
      901 ggagggctgt ctgcttccca gaagtccaag gacacaaatg ggtgagggga gagctctccc
      961 catagctggg ctgcggccca accccacccc ctcaggctat gccagggggt gttgccaggg
     1021 gcacccgggc atcgccagtc tagcccactc cttcataaag ccctcgcatc ccaggagcga
     1081 gcagagccag agcaggttgg agaggagacg catcacctcc gctgctcgca agctttattg
     1141 cggtagttta tcacagttaa attgctaacg cagtcagtgc ttctgacaca acagtctcga
     1201 acttaagctg cagaagttgg tcgtgaggca ctgggcaggt aagtatcaag gttacaagac
     1261 aggtttaagg agaccaatag aaactgggct tgtcgagaca gagaagactc ttgcgtttct
     1321 gataggcacc tattggtctt actgacatcc actttgcctt tctctccaca ggtgtccact
     1381 cccagttcaa ttacagctct taaggctaga gtacttaata cgactcacta taggctagcc
     1441 tcgagctcgg atccaccacc atgttcaaaa atcttatttg gctgaaggaa gttgatagca
     1501 cccaggagcg gcttaaggaa tggaatgtct cctatggcac tgcactcgtc gccgatagac
     1561 agaccaaagg tagaggcggt ctcggacgga agtggttgag tcaggagggc ggtctttact
     1621 tctctttctt gttgaatcca aaagagtttg aaaatttgtt gcaacttcca ctggtcttgg
     1681 gactctccgt gagcgaggca ctcgaagaga ttacagagat accatttagt ctgaagtggc
     1741 ccaacgacgt ttattttcag gagaaaaagg tttcaggggt cctttgtgag ctcagtaagg
     1801 acaagctgat agtgggaatc ggtataaacg tgaatcaaag agagatccct gaagagatca
     1861 aggatcgggc cacaacactt tatgaaatca ctggaaagga ttgggacaga aaggaggtct
     1921 tgctgaaggt tctcaagcga atttccgaaa atctcaagaa atttaaggaa aaatccttta
     1981 aagagttcaa aggaaaaatt gagtcaaaga tgttgtacct tggagaggag gttaaactct
     2041 tgggagaagg caagattacc gggaaacttg tgggcctgag tgaaaagggc ggtgctttga
     2101 tacttacaga ggaaggcata aaggagatac tgtccggcga atttagcttg cgacggtcaa
     2161 gcttaggtgg aggaggttct ggaggcggtg gaagtggtgg cggaggtagc ggtggaggag
     2221 gttctatggt gtctaaggaa ttcatgttca aaaatcttat ttggctgaag gaagttgata
     2281 gcacccagga gcggcttaag gaatggaatg tctcctatgg cactgcactc gtcgccgata
     2341 gacagaccaa aggtagaggc ggtctcggac ggaagtggtt gagtcaggag ggcggtcttt
     2401 acttctcttt cttgttgaat ccaaaagagt ttgaaaattt gttgcaactt ccactggtct
     2461 tgggactctc cgtgagcgag gcactcgaag agattacaga gataccattt agtctgaagt
     2521 ggcccaacga cgtttatttt caggagaaaa aggtttcagg ggtcctttgt gagctcagta
     2581 aggacaagct gatagtggga atcggtataa acgtgaatca aagagagatc cctgaagaga
     2641 tcaaggatcg ggccacaaca ctttatgaaa tcactggaaa ggattgggac agaaaggagg
     2701 tcttgctgaa ggttctcaag cgaatttccg aaaatctcaa gaaatttaag gaaaaatcct
     2761 ttaaagagtt caaaggaaaa attgagtcaa agatgttgta ccttggagag gaggttaaac
     2821 tcttgggaga aggcaagatt accgggaaac ttgtgggcct gagtgaaaag ggcggtgctt
     2881 tgatacttac agaggaaggc ataaaggaga tactgtccgg cgaatttagc ttgcgacggt
     2941 cagctagcgc ttacccttac gatgtaccgg attacgcata gggatccaat tcgcttatcg
     3001 ataatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg
     3061 ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc
     3121 gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt
     3181 tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca
     3241 ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc
     3301 ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc
     3361 tgttgggcac tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc
     3421 tcgcctatgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc
     3481 tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc
     3541 ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg catcgatacc
     3601 ggaattctgc agatatctag agtcgacccg ggcggccgct tcgagcagac atgataagat
     3661 acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg
     3721 aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca
     3781 acaacaattg cattcatttt atgtttcagg ttcaggggga gatgtgggag gttttttaaa
     3841 gcaagtaaaa cctctacaaa tgtggtaaaa tcgataagga tcttcctaga gcatggctac
     3901 gtagataagt agcatggcgg gttaatcatt aactacaagg aacccctagt gatggagttg
     3961 gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga
     4021 cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagcct taattaacct
     4081 aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt
     4141 aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc
     4201 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc
     4261 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     4321 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     4381 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     4441 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     4501 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     4561 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     4621 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     4681 atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     4741 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     4801 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     4861 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     4921 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     4981 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     5041 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     5101 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     5161 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     5221 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     5281 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     5341 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     5401 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     5461 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     5521 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     5581 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     5641 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     5701 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     5761 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     5821 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     5881 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     5941 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     6001 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     6061 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     6121 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     6181 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     6241 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     6301 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     6361 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     6421 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     6481 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     6541 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     6601 gcagcgagtc agtgagcgag gaagcggaag
//